Curiosity over the origin of a naturally occurring
albino bud sport on a plant of Audie Murphy, on the
part of my physicist friends, Philip Livdahl and Harlan Zodtner, led to the consideration of experiments
to induce mutation. Since studies -on the effect of
garmna radiation were already underway at Brookhaven, neutron radiation was an obvious line of attack. The following experiments, begun in the fall of
1957 and continuing until 1959, have given such unusual results as to be worth reporting.

Materials and Methods

Rosebuds of the vigorous Grandiflora rose, Queen
Elizabeth, a red Hybrid Tea H56024/39, and Rosa multiflora were cut from bud sticks and placed on
wet filter paper, 50 in each Petri dish. Except for a
control groupof about 100 buds left on bud sticks in
Experiment 1, this method of exposing buds was used
throughout. The following five experiments were
performed:

1. In the fall of 1957, Philip Livdahl, then at Lawrence Radiation Laboratory in Livermore, California
(now at Argonne Laboratory near Chicago), used
the A-48 linear accelerator bombarding a copper target with 7.5 Mev deuterons. The approximate
radiation intensity at the main axis of the beam was
estimated to be about 400 rad, with neutrons emitted
at a wide angle. Two Petri dishes, each with 50 Queen
Elizabeth buds were placed one immediately behind
the other. A small bundle of bud sticks having about
100 buds of Queen Elizabeth was placed as near as
possible to these Petri dishes as a control, to study
possible shielding action of bud stick tissue. All the
exposed buds and 50 control unexposed buds were
budded into R. multiflora understock the same afternoon immediately following exposure.

2. On March 14, 1958, 50 buds of Golden Rapture
#5 were exposed to thermal neutrons from a "water
boiler" reactor receiving approximate 1000 rad and
budded into R. multiflora un-derstock in the greenhouse.

3. On March 18, 1958, four Petri dishes, each with
25 buds of Golden Rapture #5 were exposed to 400,
600, 1500, and 3000 rads respectively of 14 Mev neutrons from a Cockcroft-Walton accelerator. These
were neutrons resulting from deuterons accelerated
to 500 kev bombarding a tritium target. The buds
were immediately budded into R. multiflora understock in 4" pots in the greenhouse.

*Paper presented at the Fifteenth Annual Convention of
the American Scientific Affiliation, Seattle, Washington, August, 1960.

**Dr. Lammerts received his Ph.D. in Genetics from the
University of California and is Director of Research for
Germains Seed Company, Livermore, California.

4. On April 23, 1959, 50 buds of a red Hybrid Tea
H5 5024/39 were placed 11 centimeters from the target of a Van de Graaff accelerator and exposed to
about 90 rads of 15 Mev neutrons obtained by the
accelerated deuterons impinging on a tritium target.
S,imilarly 4 other petridishes of 50 Queen Elizabeth
buds each, were exposed to 90 and 120 rads, and 2 at
180 rads. As a check to test the effect of placing one
Petri dish behind the other, an upper dish at 9 centimeters away was exposed to 120
rad. All were budded in R. multiflora understock soon after exposure;
i.e., the same afternoon.

5. Finally on July 9, 1959, 3 Petri dishes (150
buds) of H56024/39, 1 Petri dish (50 buds) of
Queen Elizabeth, and 50 buds of the single 5-petaled
white R. multiflora understock were exposed at 9%
cm from thetarget to 15 Mev neutron radiation obtained by using a Van de Graaff accelerator. As a
c eck on the "Protective effect" of Petri dishes a
,, triple piggyback" group of 3 Petri dishes, each with
50 buds of Queen Elizabeth, were placed one above
the other so that the dish closest to the target (8 cm)
received 500 rad of neutron radiation. These were
all budded into R. multiflora understock the same
afternoon following radiation exposure. The R. multiflora, single Petri dish of Queen Elizabeth, and all
3 of the H56024/39 Petri dishes at 9Y2 cm. received
about 350 rad of neutron radiation. The third Petri
dish of the "triple piggy back" group received about
28D rad. One of the H56024/39 dishes was removed
after 180 rad of neutron radiation.

Results

Before giving results it should be explained that
Queen Elizabeth is a hybridof the Hybrid Tea rose,
Charlotte Armstrong x Floradora, a Florabunda.

Experiment 1-The most striking result of this
experiment was the almost complete lack of mutations or even bud retardation from buds left on bud
sticks when exposed. By comparison the 50 buds in
the Petri dish nearest the target were all retarded and
slow to start and very deformed as to foliage and leaf
appearance. Gradually many of -them became more
normal by growth from buds on deformed stems, but
even these shoots were abnormal, having istrap"
leaves and segments of heavily pigmented tissue.
Finally by the spring of 1958, most of the plants had
normal shoots which ",took over" the growth. The
original deformed branches continued to grow, and
buds taken from them showed a wide range of variation. About 20 of the plants, however, remained completely changed, both as to color, form, number of
flower petals, and appearance of plant and leaves. A
thick large rugose leaf type was obtained so unlike

Queen Elizabeth that one would never suspect its relationship by induced mutation. Equally unusual was
a very narrow strap leaf type, having thin long,
pointed buds. Several had flowers about the size of
Floradora with 50-60 petals as compared to 20-25 in
Queen Elizabeth and were light scarlet in color, All
were weaker in growth than Queen Elizabeth and
one was so similar, except for its dwarf habit, as to
be of possible commercial value, since some gardeners
object to the remarkably vigorous growth of Queen
Elizabeth.

Even more interesting was the unusual fact that
plants from buds in the petri dish above the one next
to the target were normal, except for one possible
color variation to light scarlet. The buds were but
little retarded, as compared to the control buds not
exposed to any radiation.

Experiment 2 may
briefly be stated as negative, in
that no mutations were observed following exposure
to thermal neutron radiation.

Experiment 3
was discouraging in that the two
most desired mutations, i.e., increased petal number
and darker yellow color, were not obtained. Occasional changes, such as reduction of petal number to
5-6 from the usual 20-25 (2 plan~ts), and one plant
with slightly narrower leaves were observed in those
radiated at 3000 rad. These changes are valueless
commercially. Evidently the genetic variability potential of Golden Rapture #5 was not great. Possibly this may be due to the fact that it carries at
least 2 factors for pale yellow (YY). Hence, though
genetically carrying recessive factors for deep yellow (yy), both dominant factors would have to be inactivated to allow expression of recessive deep yellow
as 'hoped for.

Experiment
4-As the buds of those exposed to
lower dosages of the high energy neutron radiation
of this experiment began growing, it seemed at first
that, even at this low level, we had variability coinparable to that of Experiment 1. The following is a
summary of observations made June 9, 1959, followed
by summary of final results, as of August 18, 1960.

1. H56024/39-20-25 petaled, rose-red hybrid were radiated at 89 rad. 15 plants were normal in height, i.e., 16"-20".
7 were V-8", 5 were 2"-4", and 8 buds were just starting.
Some were of normal height with lighter anthocyanin pigmentation. One plant has flowers with only 7 petals.

8/18/60. 34 surviving plants. One very weak plant died after
transplanting. Final results were: I plant has 8-10 petals
and 2-3 petaloids, and 1 plant is very slender and weak and
12" high compared to normal pf 26"-W.

2. Queen Elizabeth-89 rads exposure. Nine were normal
16"-20", 13 were 6"-8", 20 were 1"4", 3 buds were just
starting, 2 were dormant, and 3 were dead. Some normal
plants were darker in anthocyanin pigmentation and all
showed malformation areas.

8/18/60. 22 surviving plants. All were normal Queen Elizabeth plants both as to flower and plant habit.

3. Queen Elizabeth-178 rads exposure. Ten normal plants
were 16"-20", 20 were 6"-8", 16 were V'-V, 3 buds were just
starting, 1 was dormant, and 2 buds were dead. Much greater deformity was present, even in those plants normal in
height, than among the group receiving only 90 rads. One
plant was almost black with anthocyanin. About a dozen
were sectoral chimaeras for 50-60 petals; several of these
also were light scarlet.

8/18/60. 28 surviving plants. Six were much weaker than
normal, of which 2 had abnormally small leaves and flowers.

4. Queen Elizabeth-178 rads exposure. Four were normal
16"-20", 22 were C-8", 19 were 1"-4" high, 3 were dormant,
and 4 buds were dead. Like #3, all plants were much deformed, even those normal in height.

8/18/60. 24 surviving plants. Four were much weaker
than normal.

5. Queen Elizabeth-118 rads exposure. Four were normal
16"-20", 18 were 6'4% 18 were 1"-4", 4 buds were just
starting, 4 were dormant, and I was dead. Much deformity
was present in all, seemingly more than in #3 or#4. Some
small plants with leaves were not at all like Queen Elizabeth.

8/18/60. 34 surviving plants. All quite normal in appearance.

6. Queen Elizabeth-177 rads exposure. The bottom petri
dish was nearest target at 9 centimeters. These generally
were more affected than any. None were normal in height:
15 were 6"-8" high, 24 were 1"-4!', 3 were just starting, 3
were dormant, and 2 were dead. Many, as they developed,
showed sectoral chimaeras for increased petal number, light
scarlet, and small-sized flower.

8/18/60. 33 surviving plants. One plant had most of its
leaves smaller and yellowish green. Flowers definitely were
more scarlet. A normal shoot is now growing vigorously
from the base. One plant had a thicker, leathery, dark green
foliage and a light scarlet flower. Two very weak plants
had narrow, dark green foliage. Three plants had sectoral
chimaeras, i.e., stems having flowers much more scarlet than
typical of Queen Elizabeth; one of these had over 60 petals,
beautifully imbricated. All three stems were weaker than
the rest of the plant.

At low-dosage rates evidently only a small percentage of cells in each bud mutates. As the plants
continue to grow, normal shoots "take over" and
except as noted the final result was a normal Queen
Elizabeth plant.

Generally then the many changes were transitory,
i.e., chimaeral. However, by budding from these,
some were stabilized and may have horticultural value, even though they are not as vigorous as Queen
Elizabeth.

Experiment
5, which was started on July 9, 1959,
was comparable to Experiment 4, as regards Queen
Elizabeth and H56024/39. In addition, 50 buds of
R. multiflora were exposed to 356 rads of neutrons.
Since the results are rather similar, they may briefly
be summarized as follows:

1. Queen Elizabeth-50 buds in petri dish 8 cin from target
receiving 502 rads of neutrons. Only 4 plants survived which
are now quite normal. Ten of the exposed buds started and
the plants at first were very delicate and prostrate. But
buds from these more normal and finally basal shoots were
completely normal. The original prostrate shoots are being
budded to study variability.

2. Queen Elizabeth-50 buds in petri dish 9.5 cm from
target, i.e., just above #1 and receiving 356 rads of neutron
radiation. 41 plants survived of which 5 remained much
weaker than normal. One originally prostrate plant now has
normal basal shoot. There were many sectoral chimaeras for
such qualities as:

(a) Scarlet.
(b) Small flowers similar to Floradora, having many
petals.
(c) Flowers the size of Queen Elizabeth with many petals.

Samples 4, 5, and 6,each had 50 buds of H56024/39
and received 356 rads of neutron radiation. Sample
#7, also H56024/39 received only 178 rads. There
were 49, 50, 46, and 43 surviving plants, all discouragingly normal in appearance. Occasionally
flowers with only 10-12 petals were found, but unlike Queen Elizabeth radiated buds, no flowers with
increased petal number were found.

The last experiment-petri dish #8-was 9.5 cm
from the target and received 356 rads of neutron
radiation. It contained 50 buds of a basic species,
R. multiflora, having small white flowers and only
5 petals.

Evidently this species was highly resistant to
change as there were no variations in fl~ower color,
petal number, or size. Of the 47 surviving plants,
6 are very definitely much weaker than normal and
one of these is very weak. These plants will be budded to see if this reduced vigor is a stable condition.

Discussion

Quite evidently by neutron radiation of buds at
about 4000 rad we can tremendously increase the
rate of mutation almost to the 100 per cent level in
terms of mutation per bud radiated, since all buds
showed at least sectoral chimaeras for changed tissue.

Horticulturally, the results were encouraging in
that we did get several selection with increased petal
number, having good bud form and a pleasing change
to a light scarlet or what is popularly known as "coral
pink" color. Also a dwarf type identical to Queen
Elizabeth in bud, open flower form, and color was
obtained which may have commercial value in that
some gardeners object to the extremely vigorous
growth of Queen Elizabeth (to 8 feet high in California gardens). This "buds true" in tests so far
made.

Horticulturally, the results were discouraging in
that we did not get color variations at least theoretically possible genetically. Thus, though we did get a
white variation, the leaves were adversely affected,
being semi-chlorotic, i.e., yellowish green in appearance. Possibly bud selection may segregate the color
change from the leaf change should the cell tissue
still be variable. Perhaps the situation may best be
stated by listing the variations we did not get as
follows:

2. Change to dominant S or dark scarlet such as is
characteristic of Floradora. Evidently all variations
were to the recessive s or salmon to coral pink (light
scarlet of British Horticultural Color Chart).

3. Change to the large-flower and large petal characteristics of Charlotte Armstrong. All the variations
had petals either slightly smaller to much smaller
than Queen Elizabeth.

4. Change to the long bud characteristic of Charlotte Armstrong.2 Long bud is dominant to short
bud due to a series of dominant or semi-dominant
factors.

5. Change to climbing type or more vigorous pillar
type-

6. Changes to long cutting stem with flowers occurring singly as in Hybrid Teas. All changes were
toward cluster behavior of Floradora, the Floribunda
parent.

7. Increase in leaf size. Queen Elizabeth has a
large-sized leaflet fully as large as Charlotte Armstrong. Most variations, even the large rugose one,
were toward smaller-sized leaflets.

Quite evidently most mutations were toward the
recessive type as exemplified by:

1. Decrease in size of flower coincident with increase in petal number, i.e., resulting in many smaller
petals.

2. Change to the recessive s or salmon (light scarlet appearance). These mutations were very frequent, i.e., in ratio of about 100 to 1 for change to
white.

It would seem that the dominant M factor3 was
frequently inactivated, thus allowing the recessive s
factor to show, or simultaneously the dominant S
factor was also changed to s.

Compared to Queen Elizabeth, Golden Rapture is
much more difficult to affect by radiation. Genetics
tests show definitely that it carries recessive factors (yy) for dark yellow, a much desired color. Accordingly it was hoped that neutron radiation would result in mutation to dark yellow. However, quite
evidently, much higher dosage rates must be used
and probably more buds radiated in order to inactivate both of the dominant YY factors present. Contrariwise, Queen Elizabeth has only one dominant
TNT factor which was relatively easy to inactivate, as
judged by the frequency of sectoral chimaeras for
light scarlet.

Both the red H56024/39 and Golden Rapture are
of Hybrid Tea ancestry. Unlike Queen Elizabeth
they are not Fl hybrids between a many-petaled,
small-flowered Floribunda and a large flowered Hybrid Tea. Accordingly they do not carry factors for
many petals. Hence their variability potential in this
respect is low. We know that the single, 5-petal con-

dition is recessive. As noted above, both H56024/39
and Golden Rapture mutations to 5 petals were
found, as well as intermediate conditions. Mutations
increasing the number of dominant factors for increased petalage simply did not occur.

Finally the stability of the R. multiflora species
is highly significant. Again mutations to the dominant, many-petaled condition simply do not occur.
Likewise mutations to the A factor, basic to pigment
formation, the dominant R factor for crimson red, or
the S factor for dark scarlet did not occur. Again it
is significant that there were no variations to increased flower size. R. multiflora is a semi-trailing,
climbing type of plant. Though the sample is small,
it is also significant that no mutations to upright bush
type of growth, or recurrent flowering occurred. The
conclusion is inescapable that mutations to dominant
traits are difficult to effect by radiation.

In other words the success of a radiation experiment depends, not only on dosage rate, but the variability potential of the variety used.

Biologically stated, all changes were toward weaker less viable types, certainly not comparable to
Queen Elizabeth in vigor or survival ability. In fact,
it was quite clear that as the plants developed, the
most viable tissue combination "took over" in that if
any normal unchanged cells were present, eventually
buds formed from them and they became the main
plant. In the case of the 1957 experiment, evidently
the 20 plants which remained changed and transmitted their changed traits by budding, were of
homogeneous cell type. However, as these grew from
buds, one could observe segregation of tissues and
the plants grew progressively stronger as the cells
with best gene combinations "took over" and became
buds growing into uniform-appearing but changed
plant type.

Some of these obviously differ from Queen Elizabeth by many factors. Since all are either completely
sterile or semi-sterile, it may never be possible to determine their exact genetic make-up. Crosses of
four, which showed a low percentage of apparently
good pollen back to Queen Elizabeth, do not give
much promise of setting seed.

Though it is recognized that many of the mutated
types may well be "abnormalities" involving chromosome disintegration and so not be pertinent to the
following discussion, many obviously are rather simple changes to the recessive condition. Certainly we
have here a technique by which we can observe more
,'mutations" from one group of 50 neutron-radiated
buds than usually is possible in a lifetime of looking
at several milli-on plants per year in the field run of
plants from unradiated buds.

Are then these changes the sort of thing leading to
evolutionary divergence? In this respect the results
of neutron radiation of rosebuds checks closely with
reports of other radiation work. By far the greater
majority of all mutations found are defective. Oc
casionally as in the rose experiments, some are found
which horticulturally are desirable. Even certain ones
are recorded in the literature as showing increased
resistance to some plant disease. But so far careful
inquiry has brought out that as in roses, associated
defects make the survival value of the mutant type
in a natural environment highly questionable.

It would seem then that the idea that evolution can
occur through the accumulation of mutations is simply not borne out by the ever-increasing array of experimental evidence. In fact, it becomes more and
more clear that species and even varieties are very
complexly integrated units tolerating very little "tinkering" or change. Quite obviously some species such
as the chromosomally basic R. multiflora species are
particularly resistant to change. Others have a greater variability potential.

Quite obviously the ability of species and varieties
to change as they spread over the surface of the earth
has been pushed out of all proportion into the idea
of evolution. The inability of older naturalists such as
Darwin to evaluate this variability potential of species
and varieties properly and so to mistake it for evolution or change of one species into another is quite
understandable. After all, they had no clear concepts
of genetics and even set up genetic postulates completely at variance to what we now know occurs. But
the continued interest and belief in evolution, at least
in some modified form, on the part of so many modem biologists is puzzling, since the facts of genetic
variability and mutation so clearly show that species
and varieties have such clearly defined boundaries of
variability.

Contrariwise, the idea of evolution has had a deadening effect on the minds of many, especially younger
and less imaginative research workers, since the very
concept tends to make one feel that changes come
slowly. Actually, plant breeding experience shows
that within the limits of species or variety-variability
potentials changes can be effected very rapidly. In
fact, with the development of modern tools of radiation research, we can in a few months probe the
variability range of even such normally slow reproducing species as apples, pears, and peaches. Thus by
neutron radiating buds of peach varieties and growing the budlings under continuous light,4 we can get
a crop in one year from radiated buds from which we
can see the entire range of variability potential. However, we also must be clearly alert to the fact that
once the range of variation is reached, further attempts to increase variability are a waste of time.

Conclusion

1. Data are given showing technique and dosages
use to get maximum percentage of mutations from
rosebuds by neutron radiation.

3. Horticulturally, neutron radiation of buds shows
great promise, since it so greatly speeds up mutation.

4. Biologically, all mutations are defectives, hence
any postulate of evolution by mutation is contrary to
evidence.

5. It is observed that the concept of evolution has
had a bad influence on research in that it tends to
make the scientist work on the theory that nature acts
slowly and mutational changes occur slowly. Hence
he tends to limit himself as to possibilities of getting
results rapidly.

ACKNOWLEDGMENT

The author wishes to acknowledge the advice and help
of Philip Livdahl, Howard Tewes, and Harlan Zodtner
in planning and executing the radiation of the rosebuds
in these experiments.